GSM3424036: 293T Med26 ChIP Rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
anti-MED26 H-228, Santa Cruz sc-48776 X
Sequenced DNA Library
For RNA-seq, total RNA was extracted using miRNeasy Mini Kit (Qiagen #217004) and quantified using RNA 6000 Nano Kit (Agilent Technologies) in Bioanalyzer. 1µg of total RNA was subjected to oligo-dT selection or removal of rRNA (Ribo-Zero rRNA removal kit ,Illumina, #MRZH11124). ERCC spike-ins from Ambion were added to RNA prior to library preparation. For MED26 ChIP-seq, cells were crosslinked with 2 mM DSG in PBS for 30 min and then 1% formaldehyde for 20 min. For Pol II ChIP-seq, human cells and mouse cells used for spike-in control were crosslinked with 1% formaldehyde for 20 min at room temperature. Human and mouse chromatin were mixed in 10:1 ratio. Crosslinked chromatin was sonicated to generate fragments of 150-500 bp prior to immunoprecipitation. For PRO-seq, around 25 million wild type or mutant HEK293T cells were mixed Drosophila Kc167 (10:1 ratio), permeabilised, and incubated with biotin-labeled NTPs. Biotinylated RNA was hydrolyzed with NaOH, and biotin containing RNA fragments were enriched by affinity purification using streptavidin-coated magnetic beads. A 3' sequencing adaptor was ligated to the biotinylated RNA, the 5'cap was removed, 5'OH was generated by base hydrolysis and converted to 5′ phosphate by treatment with T4 polynucleotide kinase, a 5′ sequencing adaptor was ligated to RNA. RNA was purified to enrich for biotinylated fragments with adaptor at both ends and reverse transcribed. Detailed methods are in PRO-Seq, Mahat et al Nat Protoc, 11:1455-76; doi: 10.1038/nprot.2016.086. Ribo-depleted RNA seq libraries were prepared using TruSeq Stranded Total RNA kit (Illumina, #20020596) following manufacturer's instructions. PolyA-selected RNA-seq libraries were prepared using TruSeq Stranded mRNA kit (Illumina, #20020594) following manufacturer's instructions. Both TruSeq kits add Illumina-compatible adapter and indexes for sequencing. Libraries were checked for quality (Bioanalyzer) and quantity (Qubit). Equimolar concentrations of libraries were pooled, requantified and sequenced as 50bp single reads on the Illumina HiSeq 2500 instrument. ChIP-seq libraries were prepared using KAPA HTP kit (Roche, #KK8234) and stock adapters by Bioo Scientific NEXTflex DNA barcodes with 15 cycles of PCR. Libraries were purified using Agencourt AMPure XP system and quantified using Bioanalyzer and Qubit fluorometer. Post amplification size selection was performed on all libraries using Pippin-prep (Sage Science) instument. Equimolar concentrations of libraries were pooled, requantified and sequenced either as single reads on the Illumina HiSeq 2500 platform (polyA-selected RNA-seq and MED26 ChIP-seq) or as single read High Output on Illumina NextSeq 500 platform (ribo-depleted RNA-seq and Pol II ChIP-seq). PRO-seq libraries were generated essentially as described in Mahat et al., 2016 (Nat Protoc, 11:1455-76; doi: 10.1038/nprot.2016.086). Primers RPI4, RPI5, RPI6 and RPI7 were used to barcode cDNAs from 293T_WT_Rep1 (Wild Type, replicate 1), 293T_WT_Rep2 (Wild Type, replicate 2), 293_D2G8-MED26-MUT_Rep1 (Mutant MED26-D2G8, replicate 1), and 293_D2G8-MED26-MUT_Rep2 (Mutant MED26-D2G8, replicate 2) respectively.